This invention describes a method and system for the consistent and effective encapsulation of viable biological material (e.g., individual living cells, cell clusters, or organ tissue) with a polymeric coating material.
Transplantation of organ tissue (e.g., pancreatic islets) into genetically dissimilar hosts has gained a significant interest in the treatment for functional deficiencies of secretory and other biological organs. Transplantation, however, generally requires the continuous use of immunosuppressive agents by the transplant recipient in order to forestall rejection of the transplanted tissue by the recipient""s immune system. Unfortunately, these immunosuppressive agents can deprive the recipient of adequate protective immune function against diseases.
A potential solution that avoids the need for such immunosuppressive agents is the encapsulation of the tissue material so as to protect the transplanted tissue from the recipient""s immune system. Encapsulation generally eliminates the need for immunosuppressive agents to prevent adverse immune system response and rejection of the implant. Encapsulation with a sufficiently semi-permeable protective barrier coating not only generally prevents an immune response, but also provides for diffusion of oxygen into the encapsulated material along with the transfer of nutrients, ions, glucose, and hormones, as well as the excretion of metabolic waste. This maintains the health of the encapsulated tissue material.
One promising approach for the encapsulation of tissue material such as pancreatic islets involves the use of coatings formed of a non-fibrogenic alginate, a gelatinous substance that can be derived from certain kinds of kelp. The islets are suspended in a viscous, liquid alginate, which is then atomized by any of a number of different arrangements into droplets of suitable size to encapsulate the islets. Once the droplets come into contact with a gelling solution, such as calcium chloride or barium chloride, a single layer alginate coating is created around the islets. Examples of this approach for creating single layer alginate coatings using an electrostatic coating process are shown in U.S. Pat. No. 4,789,550 (Hommel et al.), U.S. Pat. No. 4,956,128 (Hommel et al.), U.S. Pat. No. 5,429,821 (Dorian et al.), U.S. Pat. No. 5,639,467 (Dorian et al.), U.S. Pat. No. 5,656,468 (Dorian et al.) and U.S. Pat. No. 5,693,514 (Dorian et al.). An example for creating a single layer alginate coating using an air knife process is shown in U.S. Pat. No. 5,521,079 (Dorian et al.). A pressurized process for coating droplets is described in U.S. Pat. No. 5,260,002 (Wang) and U.S. Pat. No. 5,462,866 (Wang). Other examples for creating a single layer alginate coating using a spinning disk arrangement are shown in U.S. Pat. No. 5,643,594 (Dorian et al.) and U.S. Pat. No. 6,001,387 (Cochrum). Examples for creating a single layer alginate coating using a piezoelectric nozzle are shown in U.S. Pat. No. 5,286,496 (Batich et al.), U.S. Pat. No. 5,648,099 (Batich et al.) and U.S. Pat. No. 6,033,888 (Batich et al.)
A problem common to all of these techniques for creating single layer alginate coatings is the formation of non-encapsulated or partially encapsulated islets. Any non-encapsulated biological material or capsules that are only partially coated or that have too thin of a coating will lead to an adverse immunological response when transplanted into the recipient. One way to decrease this problem is to increase the diameter of the single coating so that the capsules have diameters in the range of 700-800 microns. Unfortunately, these large-sized capsules tend to be less effective when transplanted into a recipient because the larger diameter diminishes the ability of oxygen to penetrate completely into the interior of the capsule. It has also been found that large-sized capsules tend to increase the potential for macrophage attack by the recipient""s immune system and can limit the potential transplantation sites as compared to smaller-sized capsules.
It has been discovered that encapsulation of tissue material such as pancreatic islets with a second coating of a cross-linkable polymer can provide substantially complete coverage of the islets in order to minimize or eliminate the possibility of adverse immune reactions, while at the same time providing a capsule having a dimension on the order of 400-500 microns. The multiple coatings of the individual capsules containing the core of living tissue serve as an additional means for assisting in the resistance to chemical, mechanical, or immune destruction by the host. The smaller-sized capsule is believed to permit oxygen to better permeate into the interior of the capsule as oxygen can normally permeate up to about 200-250 microns into encapsulated tissue material.
U.S. Pat. No. 5,470,731 (Cochrum) and U.S. Pat. No. 5,531,997 (Cochrum) describe a double layer coating for tissue that comprises a first layer of a gellable organic polymer and a cationic polymer and a second water-soluable, semi-permeable layer chemically bonded to the first layer. U.S. Pat. No. 6,020,200 (Enevold) describes a dual layer coating having a stabilized outer layer formed of a cross-linked polmer matrix. U.S. Pat. No. 5,227,298 (Weber at al.) describes a double walled alginate coating. U.S. Pat. No. 5,578,314 (Cochrum et al.) teaches such a method for applying multiple layers of alginate onto biological material (e.g., pancreatic islets). In this method, a first layer of a multiple layer alginate coating is applied using a solution of an alginate containing a high ratio of guluronate to manuronate, and the second layer is applied using a solution of an alginate containing a high ratio of mannuronate to guluronate. U.S. Pat. No. 5,876,742 (Cochrum et al.) teaches a multiple layer alginate coating where an intermediary halo layer of a soft gel is formed between the inner and outer alginate coating layers.
While the use of multiple layer alginate coatings solves many of the problems associated with single layer coatings, the existing techniques for generating such multiple layer alginate coatings are not well suited to large scale manufacturing systems that can consistently and reliably produce large amounts of encapsulated material. In order to obtain amounts of encapsulated islets, for example, necessary for a single human transplantation procedure, as many as 500,000 to 1,000,000 encapsulated islet equivalents (one islet equivalent is equal to a cell cluster of islets having a diameter of 150 microns), or at least 8000 islet equivalents per kilogram of body weight may be required.
Several problems with the existing techniques have generally prevented the large-scale manufacture of encapsulated islets to meet these needs. First, the existing techniques tend to generate a very large number of empty capsules or xe2x80x9cblanksxe2x80x9d. While such blanks can be created in either the first coating process or the second process, the problem is most noticeable where a droplet is produced during the second coating process that does not contain an islet. Second, the existing techniques also tend to create encapsulated islets in which multiple single coated islets either stick together during the coating process or end up with more than about ten islets being contained within the same second coating encapsulation, conditions which are referred to as xe2x80x9cclumpingxe2x80x9d. When clumping occurs during the coating process, the entire batch of capsules being processed can be destroyed. Single-coated capsules can bind together into clumps that are subsequently coated a second time during the encapsulation process. Depending upon the number of single-coated capsules in a particular clump, the coated clumps do not function as effectively as a double-coated capsule containing only one or up to four single-coated capsules, most likely because the size of the resultant capsule of clumped single-coated capsules is too large.
Most importantly, the entire encapsulation process for biological materials is a time and stress sensitive process. The longer that living tissue or cells are exposed to the process or the stresses created by the encapsulation process, the less viable and effective the resulting encapsulated tissue material will be. Moreover, it has been discovered that encapsulated islet cells, for example, have a limited viability of only a few days in cell culture, both prior to encapsulation and after encapsulation and prior to transplantation. The existing techniques for double alginate coating have been limited to processing relatively small batches of cell clusters, on the order of only tens of thousands of islets per batch. Even with these small batches, the process can take several hours, exposing the cells to stress during the entire period of the coating process. The ability to process only small batches of islets requires the encapsulation process to be repeated numerous times in order to obtain the requisite number of islets for a single transplantation, thereby potentially extending the time that the encapsulated islets must be maintained in cell culture prior to transplantation. Small batches are also inefficient in that a certain number of the islets will be lost during each process. When small batches are processed, the number of islets lost represents a larger percentage of the total islets processed than if larger batches could be processed.
Although multiple layer alginate coatings for encapsulating tissue material have offered promise as a potential alternative for protecting tissue implants without the use of immunosuppressive agents, the existing techniques are not well-suited for the large-scale manufacturing required to generate the necessary volume of encapsulated tissue material in a short period of time for a successful transplant operation. It would be desirable to provide for a manufacturing method and system for the consistent and effective encapsulation of large batches of biological material that can overcome these limitations.
A method and system for the consistent and effective encapsulation of large batches of biological material applies an electrostatic charge to capsules having a first layer coating prior to creating a second layer coating so as to singulate and separate the single coated capsules during a mixing process prior to the second layer coating process. The single layer coated capsules are suspended in a liquid carrier medium for purposes of applying the electrostatic charge. Preferably, the liquid carrier medium is low viscosity and physiologically balanced to the biological material. The electrostatically charged carrier medium with the single layer coated capsules is then introduced into an alginate solution as part of a mixing process performed prior to the second layer coating process. Preferably, both the first layer coating process and the second layer coating process utilizes a spinning disk encapsulation apparatus having a central cup with at least one groove in an interior surface that causes droplets to exit from the center cup into an outer collection chamber containing a gelling solution in one or more relatively well-defined singulated lines.
In a preferred method of the present invention, cell clusters of biological material are suspended in a first alginate solution to form a first alginate suspension. Droplets are formed from the first alginate suspension, with at least some of the droplets containing cell clusters. The droplets are then gelled to form capsules having a first layer coating surrounding at least a portion of the cells. These single coated capsules are suspended in a liquid carrier medium that is preferably low viscosity and physiologically balanced to the biological material to form a carrier suspension. An electrostatic charge is applied to the carrier suspension prior to introducing the carrier suspension into a second alginate solution to create a singulated flowstream of the carrier suspension containing the single-coated capsules. As part of the mixing process, the second alginate solution is agitated by a vortex mixer as the singulated flowstream of the carrier medium containing the single-coated capsules is introduced into the second alginate solution to create a second alginate suspension. As a result of the process of applying the electrostatic charge and agitating the second alginate solution, the capsules do not clump as they are introduced into the second alginate solution to create the second alginate suspension. After this mixing process is complete, the second alginate suspension containing the separated single-coated capsules are formed into droplets and then gelled to form a second layer coating around the capsules.
In a preferred system of the present invention, a series of apparatus and processing techniques are arranged to effectively and consistently encapsulate biological material. The system includes a system for atomizing a first alginate suspension containing cell clusters of the biological material in a first alginate solution to form droplets, the majority of the droplets containing at least one cell cluster. The droplets are gelled in order to form single-coated capsules having a first layer coating surrounding at least a portion of the cell clusters. The single coated capsules are placed in a liquid carrier medium for the purpose of applying the electrostatic charge prior to the second layer coating process. Preferably, a conductive collar is used for applying the electrostatic charge to the liquid carrier medium containing the single-coated capsules. As the carrier medium containing the single-coated cells is introduced into a second alginate solution to create a second alginate suspension, the electrostatic charge separates the single-coated capsules in a fluid stream of the carrier medium. The second alginate solution is agitated by a vortex mixer as the carrier medium is introduced. Together, the electrostatic charge and the agitation of the second alginate solution prevent the single-coated capsules from clumping together in the second alginate solution as the second alginate suspension is formed. After this mixing process is complete, a system for atomizing and gelling the separated capsules in the second alginate suspension is used to form a second layer coating around the capsules.
Preferably, the system for atomizing and gelling droplets to create both the first and second layer coatings is a spinning disk encapsulation apparatus having a center cup into which droplets of the appropriate alginate suspension are introduced and an outer collection chamber containing a gelling solution for gelling the droplets of the alginate suspension so as to form a layer coating around the capsules. In order to achieve more effective operation for the coating process, the center cup preferably includes at least one groove defined on an inner wall of the center cup. When the droplets of the alginate suspension are introduced into the center cup, the grooves cause the droplets to travel from the center cup into the outer collection chamber in one or more relatively well-defined singulated lines exiting from the center cup. A singulated line is a line of moving liquid and/or droplets that does not mingle with other lines. The grooves in the central cup provide the ability to control the location of the singulated lines of droplets, as well as the uniformity of the size and shape of the droplets created by the spinning disk encapsulation apparatus.
The present invention overcomes the problem of clumping that has traditionally been associated with scaling up the coating of biological material and reduces the problems of blanks created in the second coating process. The challenge of successfully mixing and double coating living tissue in large quantities without damaging the tissue is complex. Single coated capsules suspended in a second alginate solution behave somewhat like tapioca beads in pudding. Mixing or stirring with mechanical mixing techniques to disperse the capsules in the second alginate solution is either ineffective because the mixing is not vigorous, or is damaging to the tissue material because the mixing is too vigorous. If the viscosity of the second alginate solution is reduced or if the volume is increased so as to more easily mechanically mix the capsules, the effectiveness of the subsequent coating process is effected and the number of blanks produced in the droplet generation stage of the subsequent coating process increases significantly. The present invention recognizes that the way to avoid the clumping problem is to utilize a mixing process that avoids clumping in the first place by singulating and separating the single-coated capsules such that the capsules do not touch one another as they are introduced into the second alginate solution.
Specifically, the present invention provides a method and system for the effective and consistent encapsulation of viable (i.e., living or physiologically active) clusters of biological material (preferably, pancreatic islets also known as islets of Langerhans) with a polymeric material preferably, a biocompatible semi-permeable alginate) to form a gelled capsule, which can be transplanted into genetically dissimilar hosts. At least two layers of polymer (e.g., alginate) are applied. With the current invention, the failures and defects in the previously described methods are eliminated and it is possible to produce high quality double layer capsules in high volumes, while minimizing the processing time and stress for the biological material being encapsulated. Unlike existing double layer alginate encapsulation techniques, the present invention allows for double layer encapsulation of all of the cell clusters from a donor organ as part of one large-scale batch process. In contrast to existing techniques which typically produce blanks during the second coating process at a ratio of at least 10:1 to encapsulated cells, and more commonly at a blank ratio of 50:1, the present invention produces blanks during the second coating process at a ratio of less than 10:1 and more typically at a blank ratio of 6:1 or better.
One embodiment of a method of the present invention includes providing a composition that includes a liquid carrier medium and polymeric capsules that include biological material in a first polymeric coating; applying an electrostatic charge to the composition of carrier medium and polymeric capsules; producing a flowing stream of droplets of the composition, wherein a relatively high proportion of the droplets include the polymeric capsules; and introducing the flowing stream of droplets directly into a second polymeric coating composition to form a suspension. These steps form the electrostatic mixing process described in greater detail below. Preferably, the method subsequently includes atomizing and contacting the suspension with a gelling solution to gel the second polymeric coating composition and encapsulate the polymeric capsules having a first polymeric coating in a second polymeric coating. This process can be used to provide one or more polymeric coatings to biological material by repeating the steps of applying an electrostatic charge, introducing the flowing stream of droplets into a polymeric coating composition to create a suspension, and atomizing and contacting the suspension with a gelling solution.
This step of introducing the single-coated material (i.e., single-coated capsules, which are the polymeric capsules that include biological material in a first polymeric coating) into the second polymeric coating composition using an electrostatic charge is not carried out in conventional methods. Typically, in conventional methods the single-coated capsules are simply mixed with the second polymeric coating composition by mechanical means. In conventional methods, this suspension of single-coated capsules is then atomized by electrostatic charge or other means and collected in a gelling solution (as opposed to a polymeric coating composition). The step of the present invention of dispersing the single-coated capsules into the second polymeric coating composition using an electrostatic mixing process provides significant advantage in that it reduces the aggregation and clumping of the single-coated capsules and enhances the yield of the double-coated capsules (i.e., the capsules with a first and second polymer coating).
A preferred embodiment of the present invention is used for encapsulating pancreatic islets, although the present invention is equally applicable to encapsulation of clusters of other cells ranging in size from single cells to cell clusters as large as several hundred microns. When used with pancreatic islets, the carrier medium is a physiological saline solution and the polymeric capsules including pancreatic islets in a first polymeric coating preferably include an alginate. An electrostatic charge at a voltage of about 1 kV to about 100 kV (preferably, 5 kV to 30 kV) is applied to the composition of the carrier medium and the polymeric capsules containing the pancreatic islets to create an electrostatic charge on a flowing stream of droplets of the composition, wherein a relatively high proportion of the droplets include pancreatic islet clusters. The flowing stream of droplets is introduced directly into a second polymeric coating composition contained in a vortex mixer to form a suspension. This suspension is then atomized and gelled by introducing the suspension into an encapsulation apparatus including a spinning disk atomizer and an outer collection chamber that includes a gelling solution to gel the second polymeric coating composition and encapsulate the polymeric capsules in a second polymeric coating, which includes an alginate.
The preferred embodiment of the spinning disk atomizer includes a central cup having an opening; a reservoir portion; at least one inner wall portion connecting the reservoir portion to the opening; and at least one groove formed in the inner wall portion. Preferably, the at least one groove extends substantially between the reservoir portion and the opening. The preferred embodiment of the spinning disk atomizer directs the path of travel of the capsules from the center cup into an outer collection chamber in one or more relatively well-defined singulated lines exiting from the center cup, thereby improving the consistency of the coating process, while minimizing damage to the biological material potentially caused by collisions between droplets exiting the center cup.